Mammalian Sec16/p250 Plays a Role in Membrane Traffic from the Endoplasmic Reticulum

Abstract

Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A 1 -like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains

“…5B, in contrast to mock-transfected cells, nocodazole treatment did not result in substantial increase in the size of Sec31A-positive or ERGIC-53-positive dots in syntaxin-18-depleted cells. Similar results were obtained for other ER exit site markers, such as Sec16A (Bhattacharyya and Glick, 2007;Iinuma et al, 2007;Watson et al, 2006) and p125 (Shimoi et al, 2005). …”

“…Similar results were obtained for other ER exit site markers, such as Sec16A (Bhattacharyya and Glick, 2007;Iinuma et al, 2007;Watson et al, 2006) and p125 (Shimoi et al, 2005).…”

“…For this purpose, we performed two consecutive transfections with Syn18(390) and the plasmid encoding VSVG-GFP, as described by Iinuma et al (Iinuma et al, 2007). At the nonpermissive temperature, VSVG-GFP accumulated in smooth ER patches of Syn18(390)-transfected cells (supplementary material Fig.…”

“…Recent studies have identified several proteins participating in the organization of ER exit sites. These include yeast and mammalian Sec16 (Bhattacharyya and Glick, 2007; Connerly et al., 2005;Iinuma et al, 2007;Watson et al, 2006), mammalian p125 (Shimoi et al, 2005) and Drosophila p115 (Kondylis and Rabouille, 2003), all of which are peripheral membrane proteins that accumulate at ER exit sites. In the present study, we showed that syntaxin 18 is required for the organization of ER exit sites in mammalian cells.…”

BEACHcombing for α-granules

“…It interacts with COP II coat components Sec23A, and Sec13. 4 Given the localization of Sec16a, its interaction with Nbeal2 implies an early role for the BDCP, perhaps in granule cargo sorting at the ER of megakaryocytes. Because Nbeal2 is largely cytoplasmic, it is possible that it alternates between compartments selecting cargo at the ER and subsequently guiding its transport to nascent granules.…”